Evaluation of some plants for potential dipeptidyl peptidase IV inhibitory effects in vitro

: Objective: Dipeptidyl peptidase IV (DPP IV) is a serine amino (exo) peptidase which regulates various processes most notably plasma glucose homeostasis by cleav-ing incretin peptide hormones as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulin releasing polypeptide (GIP). Realization of the inhibition of this enzyme in controlling diabetes is one of the strategies adopted in recent years. The present study was designed to investigate the DPP IV inhibitory effects of sixteen plant having antidiabetic property in aqueous extracts in correlation with their protein content. Methods: I n vitro DPP IV inhibition was evaluated by the specific inhibitory activity of plant aqueous extracts prepared without and with heat (60°C) treatment. Results: Among the tested plants Vitis vinifera L., Artemisia dracunculus L., Prunus laurocerasus L., Rubus caesius L. and Olea europaea L. extracts showed DPP IV inhibitory activity with respect to IC 50 values of 0.04-0.09 mg protein/ml. Kinetic analysis indicated that the inhibitor potency of A. dracunculus extract was stronger than the other extracts. Conclusion: The present study is the first report on screening and preliminary characterization of DPP IV inhibitory activity in aqueous extracts of selected antidiabetic medicinal food. This study could provide a new insight into DPP IV inhibitors from plants that could be useful for treatment of Type 2 diabetes.


Introduction
Type 2 diabetes (Non-insulin dependent diabetes mellitus: NIDDM) is a chronic metabolism disorder characterised by insulin resistance or the abnormal secretion of insulin [1]. Type 2 diabetes mellitus (T2DM) is one of major causes of morbidity all over the World [2]. Currently, 150 million people worldwide are considered to be diabetic and this number is expected to rise to 300 million in year 2025 [3]. The long term manifestation of this disease can result in the development of vascular disorders, such as retinopathy, neuropathy and angiopathy [4,5]. Numbers of alternative therapies are currently under development. One such approach is the inhibition of dipeptidyl peptidase IV, the major enzyme degrading the incretins in vivo [6]. Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) hydrolyses biologically active peptides that control critical functions, such as immune response, particular T cell activation, signal transduction and T cell proliferation and metabolic homeostasis [7,8]. The discovery of the role of enzymes in most important diseases such as Type 2 diabetes accelerated the design of potential pharmaceutical agents for the treatment of Type 2 diabetes mellitus. DPP IV rapidly inactivates the incretin hormones; glucagon like peptide-1 (GLP-1) and glucose dependent insulinotropic polypeptide (GIP) which serve as important prandial stimulator of insulin secretion and regulators of blood glucose control [9,10]. Inhibitors of the dipeptidyl peptidase IV reversibly block DPP IV mediated inactivation of these incretin hormones resulted in glycemic control. Because of this fact recently researchers focused on the development of DPP IV inhibitor compounds as a major new class of oral antidiabetic agents. Several classes of DPP IV inhibitors have been progressed in clinical development [11][12][13]. Some of them are dipeptide analogs of the natural substrates, mimicking their transition state. Inhibitors from natural sources have some advantages in comparison with those of synthetic origin such as low toxicity, high stability in physiological conditions and a wide variety of chemical structures for the design of new drugs [14]. In recent years many plants were screened for their antidiabetic potential [15]. The constituents that come under the category of polysaccharides, peptides, alkaloids, glycopeptides, triterpeniods, amino acids, steroids, xanthene, flavonoids, lipids, phenolics, cumarins, iridoids, alkyl disulphides, inorganic ions and guanidines were reported to have antidiabetic activity upon variety of mechanisms [16]. Among these, methanolic extracts of Mangifera indica L. leaves was tested in vitro for DPP IV inhibitory activity [17]. However there has not been a conducted research for screening of aqueous plant extracts capable of inhibiting dipeptidyl peptidase activity. In the present study; we evaluated the screening and preliminary characterization of DPP IV inhibitory activity in aqueous extracts of selected traditional antidiabetic medicinal plants. Most of the plants tested in this study are part of dietary component, so there is less possibility of side effects caused by these plants.

Materials and Methods
All of the chemicals used were of molecular grades and obtained from commercial suppliers. Gly-Pro-p-Nitroaniline was purchased from Bachem (www.bachem.com).

Plant materials
Sixteen plants (Scientific, English, Turkish names and the parts used are listed in Table 1) were purchased from local markets in Izmir between July and September and then were identified by Dr. S. Gokhan Senol an expert on plant systematic at the Faculty of Science, Ege University Izmir.

DPP IV preparation from sheep kidney
Sheep kidneys from slaughterhouse of Tansas, Izmir, Turkey were homogenized using freeze-thaw under liquid nitrogen. Following extraction with 0.25% sucrose containing 50 mM Tris buffer, pH 8.0, the crude enzyme preparation was centrifuged at 4°C, 10000 g for 30 min. The supernatant was kept at -80°C until use. This study was approved by the Animal Research Ethical Committee of the Medical School, Ege University, Izmir, Turkey (2008 -40).

Protein determination
The protein concentrations were determined according to the micro Bradford assay [18] using bovine serum albumin as standard.  [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] were prepared as follows: Seed of plants used were coarsely powdered previously and then were homogenized in distilled water (0.5g/ml) at room temperature. Fresh fruit and leaves of plants used were homogenized in same conditions. Homogenates were centrifuged at 10000 g for 20 min and extracts were lyophilized (I). Second series (II) of plant extracts were also prepared by heating the extract for 30 min, at 60°C as was stated. The protein content of the lyophylizates (0.1 g/ml) were estimated and used for inhibition assays.

DPP IV inhibition and kinetic studies
Enzyme activity was monitored following an increase in absorbance at 405 nm resulting from the cleavage of the substrate Gly-Pro-pNA by DPP IV to release p-nitroaniline (pNA) in 96 well plates. Briefly, kidney DPP IV diluted in 0.1 M Tris buffer, pH 8.0 was incubated at 37°C for 15 min without and with appropriate amounts of plant extracts (0.025-0.01 mg protein/ml) in total volume of 100 µl. Finally 100 µl of substrate solution was added and absorbance was measured over 15 min at 405 nm in a microplate reader (Multiskan FC, Thermo Scientific). Controls without plant extracts were used as reference. For all tests the inhibition assay was performed in triplicate. One unit of specific inhibitory activity was defined as the amount of sample (in terms of protein concentration) needed to inhibit one unit of enzyme activity. Inhibitory ratio was calculated as follows: Inhibition (%)=1-(Activity sample /Activity control )x100 A standard serine protease inhibitor (PMSF from Sigma, USA) was employed as positive control. Data are expressed as mean±SEM (n=3). IC 50 values were determined by linear regression of the dose-inhibition curves in linear range and defined as the amount of the plant extracts (in terms of protein concentration) needed to inhibit the 50% of control enzyme activity. The type of inhibition was examined among the most effective plants. DPP IV activity was measured with the increasing concentrations of Gly-Pro-pNA (0.05-0.75 mM) in the absence and presence of most effective plant extracts. The type of inhibition and kinetic constants were calculated on the basis Lineweaver-Burk plots using Sigma Plot 10.0 software.

Results
In order to detect new sources of natural inhibitors of DPP IV from plant origin, we searched inhibitory activity in aqueous extracts of 16 plants which are categorized as antidiabetic and serine protease inhibitor containing antidiabetic. It should be noted that while generally cold and hot water extracts are most commonly used in the traditional method of preparing medicines in Ayurveda. Table 2 shows the results of the screening aqueous extracts of 16 medicinal plant species treated with and without heat. Depending on the preparation of crude extracts, all plant samples seemed to have potent specific inhibitory activities in terms of protein concentration except M. charantia L. Preliminary characterization of the inhibitory activity involved to study the effect of different doses on the inhibitory activity to corroborate the presence of a molecular entity characterized by a dose dependent effect. IC 50 values were calculated as a measure of inhibitory effectiveness for plants showing the most spesific inhibitory effect ( Table 2).
Thermal treatment was more effective for serine protease inhibitor containing plants especially for D. carota and V. vinifera. In the case of antidiabetic plants, thermal treatment had significant effect only for A. dracunculus. The mixed results suggested that the inhibitory molecule is resistant to heat treatment and induce the activation of target inhibitor compounds. On the other hand P. laurocerasus, R. caesius and O. europea extracts that are prepared without heat treatment had the highest specific inhibitory activity with low thermal stability. Among all obtained data indicated clearly D. carota, V. vinifera, A. dracunculus, P. laurocerasus, R. caesius and O. europea extracts could be promising for an effective DPP IV inhibitor due to their IC 50 values besides exhibiting dose-dependent inhibitory activities. O. europaea turned out to be the strongest inhibitor against DPP IV compared to positive control (PMSF). D. carota, V. vinifera, A. dracunculus, P. laurocerasus, R. caesius and O. europea were further investigated to explore the kinetic characteristics. Kinetic analysis using Lineweaver-Burk plots revealed that all of the plants that displayed high inhibitory activity did so through a partial mixed type mode of inhibition (α>β) comprising both partial competitive and partial noncompetitive components (Figure 1) that substrate bound EIS complex maintains a reduced level of catalytic activity.
Kinetic analysis indicated that Ki values for A. dracun-  (Table 3).

Discussion
Peptidases are important for many biochemical and physiological processes directly and indirectly such as digestion, fertilization, differentiation, cell signaling, wound   [17]. However, besides the extraction medium, the present study was conducted to target peptide/protein contents of the extracts. Specific inhibitory activities, IC 50 values and kinetic parameters were calculated on the basis of their protein concentrations that makes this study incomparable due to the lack of structural data. On the other hand various inhibition types such as partial and mixed type and competitive was reported for inhibitor compounds and peptides [13,36]. The Ki values and inhibitory potency we obtained allows discrimination between crude antidiabetic plant extract inhibitors for DPP IV and clarify the correlation between compound potency and stability of antidiabetic medicinal plants as well as mechanism of action of well-known antidiabetic plants by means of their DPP IV inhibitory activity. Herbs act against diabetes through various mechanisms such as insulin secretion, reducing insulin resistance, insulinomimetic effect, delaying glucose absorption, inhibitors of intermediary metabolism etc. [37]. DPP IV inhibitors are a new class of oral lowering agents for the treatment of type 2 diabetes. Although the absolute specificity of DPP IV inhibitors was not clear, several DPP IV inhibitors have shown to increase insulin secretion and reduce blood glucose level after glucose challenge [12,[38][39][40]. Thus, DPP IV inhibition represents an attractive strategy to develop antidiabetic agents.
Our findings indicate that V. vinifera, A. dracunculus, P. laurocerasus, R. caesius and O. europea extracts showed specific potent DPP IV inhibitory activity in vitro. Further studies are warranted to isolate and characterize the inhibitor compounds. Then their potential for therapeutic use in prevention and treatment of hyperglycemia and diabetes should be confirmed by in vivo studies.